Usher syndrome (USH, or just ‘Usher’) and non-syndromic retinitis pigmentosa (NSRP) are degenerative diseases of the retina. Usher is clinically and genetically heterogeneous and by far the most common type of inherited deaf-blindness in man (1 in 6,000 individuals; Kimberling et al. 2010. Frequency of Usher syndrome in two pediatric populations: implications for genetic screening of deaf and hard of hearing children. Genet Med 12:512-516). The hearing impairment in Usher patients is mostly stable and congenital and can be partly compensated by hearing aids or cochlear implants. NSRP is more prevalent than Usher, occurring in 1 per 4,000 individuals (Hartong et al. 2006. Retinitis pigmentosa. Lancet 368(9549):1795-1809). The degeneration of photoreceptor cells in Usher and NSRP is progressive and often leads to complete blindness between the third and fourth decade of life, thereby leaving time for therapeutic intervention.
Mutations in the USH2A gene are the most frequent cause of Usher explaining up to 50% of all Usher patients worldwide (±1300 patients in the Netherlands) and, as indicated by McGee et al. (2010. Novel mutations in the long isoform of the USH2A gene in patients with Usher syndrome type II or non-syndromic retinitis pigmentosa. J Med Genet 47(7):499-506), also the most prevalent cause of NSRP in the USA, likely accounting for 12-25% of all cases of retinitis pigmentosa (±600 patients in the Netherlands). The mutations are spread throughout the 72 USH2A exons and their flanking intronic sequences, and consist of nonsense and missense mutations, deletions, duplications, large rearrangements, and splicing variants. Exon 13 is by far the most frequently mutated exon including two founder mutations (c.2299delG (p.E767SfsX21) in USH2 patients and c.2276G>T (p.C759F) in NSRP patients). For exon 50, fifteen pathogenic mutations have been reported, of which at least eight are clearly protein truncating. Recently the first deep-intronic mutation in intron 40 of USH2A (c.7595-2144A>G) was reported (Vache et al. 2012. Usher syndrome type 2 caused by activation of an USH2A pseudo exon: implications for diagnosis and therapy. Human Mutation 33(1):104-108). This mutation creates a cryptic high-quality splice donor site in intron 40 resulting in the inclusion of an aberrant exon of 152 bp (pseudo exon 40 or PE40) in the mutant USH2A mRNA, and causes premature termination of translation.
The c.2299delG mutation found in exon 13 results in a frameshift causing a premature termination codon and is presumed to lead to nonsense mediated decay. Lenassi et al. (2014. The effect of the common c.2299delG mutation in USH2A on RNA splicing. Exp Eye Res 122:9-12) showed that in Usher patients the mutation leads to exon 12+exon 13 double-skipping during splicing, whereas in some patients a combination was found between exon 13 only-skip, and exon12/exon 13 double-skipping. It is not uncommon for exonic sequence alterations to cause aberrant splicing. Bioinformatics tools have predicted the c.2299delG change to disrupt an exonic splicing enhancer and to create an exonic splicing silencer within exon 13. Sequence analysis has shown that skipping only aberrant exon 13, carrying the mutation, results in removal of the frameshift mutation but also results in an in-frame link between exon 12 and exon 14. Double-skipping of exon 12 and exon 13 results in an out of frame deletion when exon 11 is linked to exon 14. Hence, whereas skipping exon 13 is desired (when carrying the c.2299delG mutation) it is preferred that exon 12 is retained.
Usher and other retinal dystrophies have for long been considered as incurable disorders. Several phase I/II clinical trials using gene augmentation therapy have led to promising results in selected groups of LCA/RP/USH patients with mutations in the RPE65 (Bainbridge et al. 2008. Effect of gene therapy on visual function in Leber's congenital amaurosis. N Engl J Med 358, 2231-2239) and MYO7A (Hashimoto et al. 2007. Lentiviral gene replacement therapy of retinas in a mouse model for Usher syndrome type 1B. Gene Ther 14(7):584-594) genes. Unfortunately, the size of the coding sequence (15,606 bp) and alternative splicing of the USH2A gene and mRNA, respectively hamper gene augmentation therapy due to the currently limiting cargo size of many available vectors (such as adeno-associated virus (AAV) and lentiviral vectors).
Despite the broad clinical potential of antisense oligonucleotide (AON)-based therapy, it is not frequently used in the vertebrate eye. AONs are generally small polynucleotide molecules (16- to 25-mers) that are able to interfere with splicing as their sequence is complementary to that of target pre-mRNA molecules. The envisioned mechanism is such that upon binding of an AON to a target sequence, with which it is complementary, the targeted region within the pre-mRNA is no longer available for splicing factors which in turn results in skipping of the targeted exon. Therapeutically, this methodology can be used in two ways: a) to redirect normal splicing of genes in which mutations activate cryptic splice sites and b) to skip exons that carry mutations in such a way, that the reading frame of the mRNA remains intact and a (partially) functional protein is made. Both methods are already successfully applied in patients with severe genetic disorders (Scaffidi and Misteli. 2005. Reversal of the cellular phenotype in the premature aging disease Hutchinson-Gilford progeria syndrome. Nat. Med 11(4):440-445; Cirak et al. 2011. Restoration of the Dystrophin-associated Glycoprotein Complex after Exon Skipping Therapy in Duchenne Muscular Dystrophy. Mol Ther 20:462-467; Cirak et al. 2011. Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: an open-label, phase 2, dose-escalation study. Lancet 378(9791):595-605; Goemans et al. 2011. Systemic administration of PRO051 in Duchenne's muscular dystrophy. N Engl J Med 364(16):1513-1522). For the USH2A gene, 28 out of the 72 described exons can potentially be skipped without disturbing the overall reading frame of the transcript, including the skip of exon 13 (while exon 12 is retained).
WO 2016/005514 discloses exon skipping AONs for the USH2A pre-mRNA, directed at skipping of exon 13, exon 50 and PE40, and/or retaining exon 12. As disclosed therein, several AONs can be used for skipping exon 13. It is therefore an objective of the invention to provide alternative and more efficient AONs that can be used in a convenient therapeutic strategy for the prevention, treatment or delay of Usher and/or NSRP caused by mutations in exon 13 of the human USH2A gene.